[Comparison of islet autoantigen-specific T cell response detected by direct enzyme-linked immunospot (ELISPOT) assay and accelerated co-cultured dendritic cells (acDCs) assay].

• W Tang
• H Y Liang
• J Yuan
• C Chao
• G Huang
• Z G Zhou
• L Yang

抄録

To investigate the effect of enzyme-linked immunospot (ELISPOT) on accelerated co-cultured dendritic cells (acDCs) and direct detection of islet full-length antigen-specific T cell response in peripheral blood of patients with type 1 diabetes mellitus (T1DM). Sixteen patients with T1DM[9 males, 7 females, mean age(28.5±9.4)years] and 12 age-and sex-matched healthy controls were selected in the Department of Metabolism and Endocrinology, the Second Xiangya Hospital between March 2012 and August 2014. The numbers of IFN-γ secreting CD4(+)T cells responding to glutamic acid decarboxylase 65 (GAD(65)), C-peptide (CP) and insulin (INS) were detected by ELISPOT-acDCs and ELISPOT-direct assays, respectively. The positive rate of islet autoantigen and associated antigen reactive T cells under different detection assays were compared. The positive rate for GAD(65), INS, and CP antigen reactive T cells detected by ELISPOT-acDCs was 1/16, 6/16 and 4/16, respectively, and T cells positive for INS in T1DM patients were higher than that in the controls (0/12) (0.024). Combining GAD(65), CP and INS-ELISPOT-acDCs detection, the positive rate for CD4(+) T cells in T1DM patients was higher than that in the controls (9/16 vs 1/12, 0.016). The positive rate for GAD(65), INS, and CP antigen reactive T cells detected by ELISPOT-direct detection was 2/16, 1/16 and 7/16, respectively, and T cells positive for CP was higher than that in the controls (1/12), but the difference was not statistically significant (0.088). Likewise, the positive rate for CD4(+) T cells was higher in T1DM patients than that in the controls by combined GAD(65), CP and INS-ELISPOT-direct detection (8/16 vs 1/12, 0.039). Compared with the ELISPOT-direct assay, the positive rate of INS antigen specific T cell response detected by ELISPOT-acDCs was higher (0.041). No statistical differences of other antigens were found between the two groups (all 0.05). Both multiple islet antigens-combined CD4(+)-ELISPOT-acDCs and direct assays could provide diagnostic value of cellular immunology for T1DM patients. The ELISPOT-acDCs assay is superior to the ELISPOT-direct assay in the detection of INS antigen-specific T cell response.

探讨酶联免疫斑点（ELISPOT）加速树突状细胞成熟的预孵育法（acDCs）和直接检测法检测1型糖尿病（T1DM）患者外周血中胰岛全长抗原特异性T细胞反应的优劣。 选取2012年3月至2014年8月中南大学湘雅二医院代谢内分泌科就诊的T1DM患者16例（男9例，女7例），年龄（28.5±9.4）岁，病程6.0（2.8~8.3）个月；另纳入年龄、性别匹配的健康对照12名；ELISPOT-acDCs法和直接检测法检测3种胰岛抗原谷氨酸脱羧酶65（GAD(65)）、C肽、胰岛素（INS）特异性干扰素-γ（IFN-γ）分泌性CD4(+) T细胞反应的斑点数，比较不同检测方法下各种胰岛抗原及联合3种抗原特异性T细胞反应的阳性比例。 ELISPOT-acDCs法检测T1DM患者GAD(65)特异性IFN-γ分泌性CD4(+) T细胞反应的阳性比例为1/16，INS反应阳性比例为6/16，C肽反应阳性比例为4/16，其中INS反应阳性比例高于健康对照组的0/12（0.024）；联合3种抗原的T细胞阳性比例为9/16，明显高于健康对照组的1/12（0.016）。ELISPOT直接法检测GAD(65)特异性T细胞反应阳性比例为2/16，INS反应阳性比例为1/16，C肽反应阳性比例7/16，其中C肽反应阳性比例高于健康对照组的1/12，但差异无统计学意义（0.088）。联合3种抗原检测T细胞反应阳性比例8/16，高于健康对照组的1/12（0.039）；与直接法比较，仅acDCs法检测下的INS特异性T细胞反应阳性比例较高（0.041）；两种方法在余各抗原间比较差异均无统计学意义（均0.05）。 多种全长胰岛抗原ELISPOT-acDCs法及直接法检测胰岛抗原特异性T细胞反应均可为T1DM患者提供细胞免疫学分型诊断价值；ELISPOT-acDCs法检测INS特异性T细胞反应的灵敏度优于ELISPOT直接检测法。.